Thrombotic thrombocytopenic purpura (TTP) is a severe thrombotic disease caused by accumulation of the endothelium-derived ultra-large VWF (ULVWF) multimers in plasma. These ULVWF multimers are hyper-reactive in interacting with their receptors. The accumulation of ULVWF is due to defects in the VWF cleaving protease that has been recently characterized as the ADAMTS 13 metalloprotease. This protease cleaves ULVWF multimers to smaller plasma form, but how and where the cleavage occurs remains completely unknown. We have recently found that the ULVWF multimers, upon secretion, adhered to the endothelial surface, forming extremely long string-like structures. These VWF strings were rapidly cleaved by the ADAMTS13 metalloprotease under fluid shear stress. Based on these results, we hypothesize that the ADAMTS13 metalloprotease cleaves the EC ULVWF multimers on the endothelial surface under fluid shear stress. To further our understanding of this proteolytic process, we propose in this application to answer two specific questions. First, how do VWF strings form and how are they anchored to endothelial ceils? We found that P-selectin antibody prevented the formation of VWF strings and hypothesize that VWF strings form as a result of ULVWF self-association through propeptides and membrane anchorage through P-selectin. Second, what are required to cleave these VWF strings by the ADAMTS13 metalloprotease? We found that the cleavage of VWF was blocked by a RGD peptide and thrombospondin, suggesting that the metalloprotease may also be anchored to the endothelial surface. We hypothesize that the metalloprotease cleaves VWF by forming a complex with an endothelial integrin (avb3) and VWF strings. We have designed experiments in this specific aim to examine these possibilities. The proposed studies are very important for understanding the cleavage of ULVWF multimers and the pathophysiology of TTP in several ways. First, they will help us to elucidate mechanisms through which the EC-derived ULVWF multimers are cleaved by the ADAMTS13 metalloprotease and; therefore, provide information as to how other members of the ADAMTS13 family may function. Although 14 members of this family of metallprotease have been characterized, functions of only two, including ADAMTS13, are known. Second, our studies may shed light on how other types of microangiopathies develop, especially HUS and TTP caused by drugs and infections. Finally, they allow us to develop a clinical assay to test the activity of the VWF-cleaving protease thus giving the ability to diagnose TTP. This assay will be fast and more physiologically relevant.